title: Expression in Escherichia coli of Y5 mutant and N-terminal domain-deleted DNA gyrase B proteins affects strongly plasmid maintenance.
abstract: Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activity in vivo. Thus, our in vivo approach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.
authors.forename: L
authors.initials: L
authors.lastname: Brino
authors.forename: M
authors.initials: M
authors.lastname: Mousli
authors.forename: P
authors.initials: P
authors.lastname: Oudet
authors.forename: E
authors.initials: E
authors.lastname: Weiss
mesh_terms: D001665:Binding Sites; D002701:Chloramphenicol / Q000494:pharmacology; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004278:DNA, Superhelical / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005784:Gene Amplification; D018628:Gene Dosage; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D004278:DNA, Superhelical; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D002701:Chloramphenicol; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D005947:Glucose